Fiber-coupled multiplexed confocal microscope

ABSTRACT

A confocal microscope system that is inherently fiberoptic compatible is described which has line scanning aided image formation. An incoherent fiberoptic bundle maps a line illumination pattern into a dispersible group of separate sources, and then remaps this confocally selected remitted light to the original line. Fibers, not confocal with the illumination, carry light to be rejected from the image back on itself upon double passing, while separate fibers carry light from non-confocal sample planes. The transformation allows efficient rejection of unwanted photons at a slit aperture. The fiber bundle and an objective lens provide a flexible probe for imaging internal tissue for pathological examination on a cellular level.

This application is a division of application Ser. No. 09/885,576, filedJun. 20, 2001, now U.S. Pat. No. 6,747,795. This application also claimsthe priority benefit of U.S. Provisional Application No. 60/215,154,filed Jun. 30, 2000. Each of these applications and each document citedor referenced in each of these applications, including during anyprosecution (“application cited documents”), and each document cited orreferenced in each of the application cited documents, are herebyincorporated herein by reference.

DESCRIPTION

The present invention relates to confocal microscopy, and relatesparticularly to confocal microscope having a parallel scanning systemcompatible with fiberoptics. The invention provides a remote probe forconfocal imaging of tissue at locations within a body, such as commonlydone with endoscopes, and thus the invention provides the advantages ofconfocal microscopy in biomedical applications by enabling access todistant and inconvenient regions.

The invention uses fiber optics, but in a way to provide significantimprovement over confocal microscopes using fibers that have beenreported, such as in which a single fiber serves as the source and thedetector pinhole.

A line scanning confocal microscope with slit aperture detection can beregarded as a form of multifocal illumination and parallel detection,where all the foci line up in one direction. Image formation requiresonly slow scanning (at 25 or 30 Hz for video rate) in the seconddirection. This approach is particularly attractive because of itssimplicity and high optical throughput. The major drawback of the linescanning approach is its relative poor rejection of unwanted photonscompared to the pinhole system.

Briefly, the present invention provides a confocal microscope in whichthe region of interest (as for example a tissue ex-vivo, or in-vivo asinside a body cavity) is illuminated via a fiber optic bundle, where thespatial arrangement of fibers at one end of the bundle is different fromthat at the other end. Such a bundle is called an incoherent fiber opticbundle. A bundle in which the spatial arrangement of fibers ismaintained is a coherent fiber bundle. The microscope may have means forscanning a laser beam from a slit providing an aperture in one directionacross, as with a slit scanning microscope, but has true two-dimensionalconfocality because of encoding the slit with an incoherent fiberbundle. The incoherent bundle effectively multiplexes and demultiplexesthe light, respectively, incident on and remitted from the region ofinterest. An important advantage of this microscope is that it canoperate at the distal end of a fiber bundle and can therefore provide aprobe on a flexible, small diameter member where it can be implementedon or as an endoscope or catheter.

It is a feature of the invention to provide an improved fiber opticconfocal microscope where the fiber effects a parallel scanningmechanism that is inherently fiberoptic compatible and that retains thesimplicity of the line scanning confocal microscope, while improving onits axial sectioning resolution. The improvement is provided by using anincoherent fiber bundle. In the bundle, the input and output fibers maybe randomly arranged, although other, specific mapping can also beemployed to obtain incoherent coupling via the fiber. A line sourceilluminates the proximal (P) end of the fiber bundle, which transformsthe line input into disperse array of fibers at the distal (D) end,spread out over the whole bundle. This set of disperse spots is imagedonto the sample by an objective lens. Remitted light (fluorescence orback scattered) from the sample is collected and imaged by the sameobjective lens back onto the fiber endface (D), from a region in thesample being viewed, which is exactly in focus, the remitted light isimaged onto the same set of fibers which carried the illumination light,and transformed back into a line at (P). For an unwanted (orout-of-focus) sample plane, each illuminated fiber at D will produce, onthe return, a smeared-out spot, which covers not just the original fiberbut a group of surrounding fibers. For spots illuminated indirectly,such as by scattered light, the same is true. Back at Plane P, thefibers which carry the “smeared-out” photons do not reassemble into aline but are spread out dispersely over the bundle. A slit aperture,placed at a plane conjugate to P, allows only light (photons) from thein-focus plane, and intentionally illuminated spots, to pass through thedetector, while rejecting most of the unwanted photons. The detectorprovides signals representing scan lines in the plane from which a 2-Dimage at that plane can be constructed.

The invention will become more apparent from a reading of the followingspecification in connection with the accompanying drawings which,briefly described, are as follows:

FIG. 1A is a view of an end of a fiber optic bundle which shows only acenter fiber thereof that is illuminated.

FIG. 1B is a view in a plane, D, across the distal end of the fiberoptic bundle of FIG. 1A when that bundle represents a coherent fiberoptic bundle.

FIG. 1C is a view in a plane, P, across the proximal end of the bundle,and showing the location of a slit aperture associated with the bundleof FIG. 1B.

FIG. 1D is a view in the plane, D, for the fiber optic bundle of FIG. 1Awhen that bundle is an incoherent fiber optic bundle according to thepresent invention.

FIGS. 2A, 2B and 2C are schematic diagrams of different embodiments ofconfocal microscope systems embodying the invention.

FIGS. 3A and 3B are exemplary images of the P and D ends which can betaken with the microscope of FIGS. 2A, 2B or 2C.

FIG. 4 are plots showing the intensity variation of the light through aconfocal slit aperture (direction z) showing the discriminating effectof the incoherent bundle by the curve having the solid dots, and withoutthe slit aperture in the curve having the open dots.

Consider FIGS. 1A to 1D and that, for clarity, a single fiber at thecenter of the bundle is illuminated (FIG. 1A). Return light from anon-confocal point is collected by the objective lens and forms andextended spot at plane D, filling a group of fibers surrounding thecentral fiber (FIG. 1B). If the fiber bundle is coherent, this group offibers will emerge at plane P with a similar pattern to that in FIG. 1Bbut surrounding the fiber of FIG. 1A. The planes P and D are at theproximal and distal ends of the bundle and are perpendicular to theoptical axis (see FIGS. 2A-C).

A confocal slit aperture placed at a plane conjugate to P and alignedwith the central fiber will reject much of the unwanted light (FIG. 1C).However, a significant fraction of the unwanted light will go throughthe slit. This is the reason why a slit aperture does not produce thesame degree of confocal rejection, as does a pinhole aperture. If thefiber bundle is now replaced by an incoherent one, then a random patternwill emerge at plane P (FIG. 1D). Rejection of unwanted light is moreefficient in this case, because the fibers carrying the unwanted photonsare now spatially separated, reducing the probability for these photonsto pass through the slit. In FIGS. 1A to 1D, the intensity ofillumination of each fiber (represented by a circle) corresponds to thedarkness of the fiber. The incoherent fiber bundle provides for anarrangement (position or location) of individual fibers at its outputend at D that are scrambled relative to the arrangement of the fibers atits input end at P either randomly, or in a prescribed pattern, and assuch, the fiber bundle does not preserve an image captured at the inputend. However, the location of each fiber of the incoherent fiber bundleat the input and output ends of the fiber may be mapped.

To form an image, the input illumination line is scanned across theplane P. The scanning can be with any suitable scan mechanism, such as agalvanometer-mounted mirror. During the scan, the output pattern atplane D is modulated in a random but deterministic fashion. On the wayback, fibers carrying photons from the sample undergo reversetransformation from D to P. In general, what comes out at plane P iscomposed of two components—a line and scattered dots, which originatefrom confocal and unwanted sample planes, respectively. The line movesacross the plane P in synchronism with the input scan line, while thescattered dots blink on and off in a random fashion. The former can bedescanned by the same scanning mirror used for the input, allowing theconfocal (in-focus) component to pass through a stationary slitaperture. The scattered dots are rejected.

Referring to FIGS. 2A, 2B, and 2C, confocal microscopes are shown havinga probe section with an incoherent fiber optic bundle FB1 and anobjective lens OL and an illuminating and imaging section. Light, suchas produced by a laser, illuminates the incoherent fiber optic bundleFB1 via optics having a slit aperture S1, and focused by objective lensOL to the region of interest, such as of tissue. The light collected bythe lens OL from the region of interest is received by the incoherentfiber optic bundle FB1 and then imaged by onto a detector, such as a CCDcamera, via optics having a confocal slit aperture S2.

The output of the stationary slit aperture rescanned onto a CCD camerayields a two-dimensional image, as is done in a bilateral scanning slitconfocal microscope, in which the CCD camera provides electronic signalsrepresentative of the image to a computer (not shown). The imageproduced in this way, however, is not an image of the sample but ascrambled image due to the action of the incoherent bundle FB1. Theoriginal image can be reconstructed by the computer operating inaccordance with decoding software if the mapping transformations of eachof the fibers from D to P is known. Alternatively, the output of theslit aperture S2 can be rescanned onto an identically mapped“incoherent” bundle FB2, which reverses the scrambling done by the firstbundle FB1 shown in FIG. 2C. The output of the second bundle FB2 canthen be imaged onto a CCD camera. The computer may be coupled to adisplay, as typical of electronic imaging confocal microscopes, to viewimages of the region of interest.

By way of a specific example of components usable in the illustratedembodiments of the invention, is a 488 mm Ar ion laser (SpectraPhysicsModel 2016). The beam expander and collimator expands and collimates theAr laser beam to a 3 mm diameter beam by lenses L1 and L2 shown in FIG.2. The beam is then focused by a 75 mm cylindrical lens L3 to a lineilluminating a slit aperture S1 (25 μm wide by 3 mm long). A lens pair(L4 and L5) with focal lengths of 75 and 150 mm, respectively, forms amagnified image (50 μm wide by 6 mm long) of the slit at plane P, whereone end of an incoherent fiber bundle FB1 is located. The fiber bundle(Edmund Scientific) FB1 maybe 30 cm long and 6.4 mm in diameter,composed of approximately 128×128 fibers with individual fiber diameterof 50 μm. The other end of the fiber bundle FB1 is placed at the backfocal plane (160 mm) of a 100X, 1.25 NA oil immersion microscopeobjective OL. Reflections at both ends of the fiber bundle FB1 may beminimized by index-matching to 6 mm thick optical windows (not shown).The objective lens OL images individual fibers down to 0.5 μm at thesample. Reflected light from the sample is collected by the objectivelens OL and coupled back into the fiber bundle FB1.

The optics of the specific embodiments are specified in Tables 1, 2 and3 for the FIG. 2A, 2B and 2C embodiments respectively. The term “f.l.”represents focal length. Note the scanning mirror M1 having front andback sides M1* and M1**, respectively, may be driven by a galvo driver,or provided by two mirrors at M1* and M1** driven synchronously, such asby a common galvo driver.

TABLE 1 Excitation beam path L1: 50 mm f.l. L2: 150 mm f.l. L3:cylindrical lens 75 mm f.l. S1: slit aperture, 25 μm wide × 3 mm longL4: 75 mm f.l. BS: beam splitter (dichroic if used in fluorescence) M1*:front side of bilateral scanning mirror L5: 150 mm f.l. W1 & W2: glasswindows index matched to fiber bundle (not needed for fluorescence) OL:objective lens Return beam path OL: objective lens W1 & W2: glasswindows index matched to fiber bundle (not needed for fluorescence) L5:150 mm f.l. M1*: front side of bilateral scanning mirror BS: beamsplitter (dichroic if used in fluorescence) L6: 75 mm f.l. S2: slitaperture, 25 μm wide × 3 mm long L7: 75 mm f.l. M1**: back side ofbilateral scanning mirror L8: 150 mm f.l.

TABLE 2 Excitation beam path L1: 50 mm f.l. L2: 150 mm f.l. L3:cylindrical lens 75 mm f.l. S1: slit aperture, 25 μm wide × 3 mm longL4: 75 mm f.l. BS: beam splitter (dichroic if used in fluorescence) M1*:front side of bilateral scanning mirror L5: 150 mm f.l. W1 & W2: glasswindows index matched to fiber bundle (not needed for fluorescence) OL:objective lens Return beam path OL: objective lens W1 & W2: glasswindows index matched to fiber bundle (not needed for fluorescence) L5:150 mm f.l. M1*: front side of bilateral scanning mirror BS: beamsplitter (dichroic if used in fluorescence) L6: 75 mm f.l. S2: slitaperture, 25 μm wide × 3 mm long DET: linear detector array or line scancamera

TABLE 3 Excitation beam path L1: 50 mm f.l. L2: 150 mm f.l. L3:cylindrical lens 75 mm f.l. S1: slit aperture, 25 μm wide × 3 mm longL4: 75 mm f.l. BS: beam splitter (dichroic if used in fluorescence) M1*:front side of bilateral scanning mirror L5: 150 mm f.l. W1 & W2: glasswindows index matched to fiber bundle (not needed for fluorescence) FB1:fiber bundle OL: objective lens Return beam path OL: objective lens FB1:fiber bundle W1 & W2: glass windows index matched to fiber bundle (notneeded for fluorescence) L5: 150 mm f.l. M1*: front side of bilateralscanning mirror L6: 75 mm f.l. S2: slit aperture, 25 μm wide × 3 mm longL7: 75 mm f.l. M1**: back side of bilateral scanning mirror L8: 150 mmf.l. FB2: identically mapped fiber bundle as FB1

With a line input at P, the pattern of illuminated fibers at D isobserved with a CCD camera. FIG. 3 shows an exemplary image of the P endof the fiber from a mirror at the position of a sample. Note that thisline pattern may be color codes with colors corresponding to films inthe bundle. Alternatively, the code may be a binary code of on and offillumination regions corresponding to the fibers in location. FIG. 3A isan image of the reflected light when the sample mirror was in focus. Asshown, most of the reflected light is carried by fibers, whichreassemble into the original slit pattern. FIG. 3B is an image at planeP of reflected light when the sample mirror was moved out of focus by ˜1μm. The brightness of the slit is greatly suppressed, while more photonsemerge from the dispersed random fibers. Some of the scattered dots thatappear outside the line in FIG. 3A are due to slight axial positionvariability. Some of the scattered dots can arise because cross talkbetween two adjacent fibers near the D end will appear as separate,scattered pixels at the P end, but cross-talk is also reduced at thecamera, by the slit.

A rectangular mask over the central column of fibers digitallyintegrates the brightness values of the pixels captured by the CCDcamera in order to provide the optical sectioning effect with a confocalslit. To show operation of the microscope, the procedure was carried outfor a series of images taken as the sample mirror was stepped throughthe focus in ˜1 μm increments. For comparison, a data set of theintegrated intensities over the entire fiber bundle was taken tosimulate the “wide field” case. A plot of the two data sets,individually normalized with respect to their peak values, is shown inFIG. 4 as a function of mirror position. The FWHM (full width at halfmaximum) of the axial response function is ˜11 μm without the slitaperture and ˜2 μm with the slit aperture.

The incoherent fiber bundle used in the example give above is onlyapproximately random in that groups of nearby fibers tend to stayclustered together from one end to the other end of the bundle.Nonetheless, substantial improvement in optical sectioning is achievedwith a slit aperture, as shown in FIG. 4. A bundle that scrambles in apre-set pattern may be preferred. Then software decoding of the imagedoes not require measurement of the fiber mapping. Such a pattern may beone that maps every row of a square matrix into a maximally separatedsquare grid that fills a matrix of the same dimension. In terms of lightbudget, a fiber bundle with a high fill factor and low numericalaperture may be preferable. The low aperture minimizes light loss die tooverfilling of the microscope objective entrance pupil. Additionally, abinary matrix pattern may be preferred for certain applications.

Variations and modifications in the above-described exemplary system,within the scope of the invention will become apparent to those skilledin this art. Thus, the description should not be taken in a limitingsense.

1. A confocal microscope comprising: at least one probe sectioninsertable into a subject for illuminating a region of interest thereofand including at least one flexible incoherent optical coupling element;an imaging section generating illumination light, and constructingimages from light remitted from the region of interest; wherein the atleast one flexible incoherent optical coupling element of the probesection transmits light between the imaging section and the probesection, and wherein the imaging section includes a second flexibleincoherent optical coupling for descrambling light received from theprobe section, whereby the confocal microscope is a remote probe forconfocal imaging of tissue at locations within the subject in place ofan endoscope.
 2. The microscope according to claim 1 wherein saidincoherent optical coupling elements are incoherent fiber optic bundles.3. The microscope according to claim 2 wherein said imaging sectioncomprises a line scanning means which scans across a proximal end of theelement.
 4. The microscope according to claim 3 further comprising aslit aperture disposed in the path of light scanned by said means acrosssaid proximal end.
 5. The microscope according to claim 2 furthercomprising an objective lens at a distal end of said element forfocusing a laser beam in said region.
 6. A confocal microscopecomprising: at least one probe section insertable into a subject havingan objective lens and a first fiber bundle coupling; a lightmanipulation section including a second fiber bundle coupling; andwherein the first fiber bundle coupling is disposed between the lightmanipulation section and the objective lens and scrambles light appliedto an end of said first fiber bundle, and wherein the second fiberbundle coupling descrambles light applied to an end thereof by saidfirst fiber bundle, whereby the confocal microscope is a remote probefor confocal imaging of tissue at locations within the subject in placeof an endoscope.
 7. The microscope according to claim 6 wherein saidfirst fiber bundle has two ends and said microscope further comprises aconfocal mask at one of said ends near said manipulating section of thefirst fiber bundle to enhance confocality.
 8. The microscope accordingto claim 7 wherein said confocal mask is a slit.
 9. The microscopeaccording to claim 6 wherein the first fiber bundle is not coherent inthat spatially, individual fibers at one of said ends of the bundle arescrambled relative to that at the other of said ends.
 10. The microscopeaccording to claim 9 wherein said individual fibers are scrambledrandomly.
 11. The microscope according to claim 9 wherein saidindividual fibers are scrambled in a prescribed pattern.
 12. Themicroscope according to claim 6 wherein the incident light forms a line.13. The microscope according to claim 6 wherein said first fiber bundlehas a distal end and light from the distal end of the first fiber bundleis imaged by said objective lens onto a sample, and remitted light fromthe sample is collected by said objective lens and coupled back into thefirst fiber bundle.
 14. The microscope according to claim 6 in whicheach end of the first fiber bundle is index matched via a windowmaterial to reduce reflection from fiber ends.